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Biomedical subjects

P Meulien

Publications and source records attributed to P Meulien.

At least 19 recordsLinked to original sources
PubMed2000

Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-HCV-positive patient.

Healthy carriers of hepatitis C virus (HCV) infection exhibit a specific antibody response against all HCV antigens, which could play a role in disease control. Generation of panels of human antibodies may permit a thorough characterization of this response and further identify particular antibodies with potential clinical value. To this effect, we have established a human phage-display antibody library from a patient exhibiting a high antibody response against HCV antigens and no clinical symptoms of disease. This library was screened against a recombinant core antigen [amino acids (aa) 1-119] produced in E. coli. Two recombinant Fab-carrying phages (rFabCs) were isolated and characterized. Both rFabC3 and rFabC14 recognize aa 1-48 on core antigen, but rFabC14 is competed out by a synthetic peptide, C(2-20) (aa 1-20), at much lower concentrations than rFabC3. In order to identify more precisely the recognition sites of these antibodies, we produced soluble forms of the rFabs (sFabs), and used them to pan a random phage-display peptide library. A single peptide sequence, QLITKPL, was identified with sFabC3, while two equally represented sequences, HAFPHLH and SAPSSKN, were isolated using sFabC14. The QLITKPL sequence was partially localized between aa 8 and 14 of core protein, but no clear homology was found for the two sFabC14 peptides. However, we confirmed the specificity of these peptides by competition experiments with sFabC14.

Adult↗
PubMed1999

Intradermal DNA immunization by using jet-injectors in mice and monkeys.

We have used spring powered jet injectors to deliver a solution of a naked DNA vaccine encoding the influenza hemagglutinin HA into the skin of mice and monkeys. We compared the immune responses induced by this needleless injection technique into the skin to the responses induced by a classical i.m. immunization. Both routes of immunization induced significant ELISA antibody titers and hemagglutination inhibition (HI) titers that were above the usual threshold values predictive of protection against influenza in mice and monkeys. In mice, both ways of immunization were equally efficient in inducing HA-specific CTL responses. Regarding antibody isotypes, the IgG1/IgG2a ratio was in favour of the IgG2a isotype for i.m. immunization and more balanced for i.d. immunization. The ability of the two injection techniques to induce immunity in mice did not correlate with transgene expression in the site of administration. In fact, local gene expression was 10-100 fold more important in the injected muscle as compared to the jet-injected skin when assessed by using the luciferase reporter system.

Animals↗
PubMed1998

DNA issues.

For many years the debate surrounding the potential danger of contaminating cellular DNA in biological products has focussed on the possibility that this DNA could give rise to tumours in the recipient host. Current knowledge of the nature and the fate of this DNA after in vivo administration allows us to conclude that this material represents a minimal risk to the patient.

Animals↗
PubMed1993

Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA.

The induction of anti-influenza cytotoxic T lymphocytes (CTL) in vivo by immunizing mice with liposomes containing messenger RNA (mRNA) encoding the influenza virus nucleoprotein (NP) is described. NP mRNA, obtained by in vitro transcription, was encapsulated into simple cholesterol/phosphatidylcholine/phosphatidylserine liposomes by the detergent removal technique. The dependence of the route of mRNA-liposomes delivery on CTL induction was studied. The CTL induced were identical to those obtained in vivo with infectious virus in terms of specificity, lysing both peptide-sensitized and virus-infected targets. Furthermore, with the same mRNA-liposome preparation, virus-specific CTL responses could be also elicited in mice of three different haplotypes each of them known to present a distinct NP peptide in an MHC-restricted fashion. The relevance of these results in the context of vaccine development is discussed.

Animals↗
PubMed1992

Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector.

The cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgII is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5' flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue-specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co-express the transgenes in hepatocytes were obtained. Hepatoma-derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.

Amino Acid Sequence↗
PubMed1990

Increased biological activity of a recombinant factor IX variant carrying alanine at position +1.

In attempts to improve the post-translational modification and processing of recombinant factor IX (FIX) we have altered the cDNA sequence encoding pre-pro-FIX using site-directed mutagenesis and have expressed the variant cDNAs in BHK21 cells using a vaccinia-virus-derived vector. We find that substitution of the tyrosine residue at +1 for an alanine increases the biological activity of the recombinant molecules 2-fold. On the other hand, substitution of the proline at -3 for a valine results in no significant change to the specific activity of the protein. Other alterations to the N-terminus of the FIX proteins, in attempts to mimic other vitamin-K-dependent proteins, result in the failure to produce a secreted polypeptide. N-terminal sequence analysis of purified recombinant molecules reveals a correlation between specific activity and the efficiency of correct pro-sequence cleavage. gamma-Carboxylation analysis of purified recombinant proteins indicates that each molecule including unmutated FIX is completely gamma-carboxylated in this system. Thus the observed increase in biological activity of FIX variants containing an alanine at position +1 is not due to increased gamma-carboxylation but, at least in part, to more efficient pro-peptide cleavage.

Alanine↗
PubMed1990

Expression in Escherichia coli of a recombinant fragment (Ile 914-Leu 1364) of human von Willebrand factor containing a collagen binding domain.

Previous studies have shown that a collagen binding domain of human von Willebrand factor (vWF) resides on a fragment named SpI obtained by digestion with Staphylococcus aureus V8 protease which corresponds to residues Gly 911-Glu 1365 of the mature plasma vWF subunit. We have subcloned a fragment of a full-length cDNA encoding vWF into an expression vector which uses an inducible lambda PL promoter. The predicted product expressed by this plasmid is a fusion protein consisting of 16 amino acids (aa) of the lambda cII protein and aa Ile 914-Leu 1364 of human vWF. This fusion protein was shown to be expressed as insoluble inclusion bodies by induced E. coli harbouring the recombinant vector and was partially purified from bacterial debris and renatured. Partially purified bacterial extract run on SDS-polyacrylamide gels contained a major band representing 50-70% of the visualized proteins and corresponded to the predicted fusion protein. This band reacted with both polyclonal antibodies against human vWF and monoclonal antibodies (MAbs) which recognize the SpI fragment of plasma vWF. The radiolabelled partially purified bacterial extract was shown to bind specifically to human fibrillar collagen types I and III. This binding, which was a function of radiolabelled ligand and collagen concentrations, did not occur on monomeric denatured collagen. It was inhibited by both unlabelled bacterial extract and plasma vWF and also by a MAb against vWF SpI, which has the property of inhibiting vWF/collagen interaction. Our data demonstrate that this recombinant vWF SpI fragment, which can be obtained in large amounts, is a useful model for localizing the epitopes of monoclonal antibodies and then for studying the mechanism of vWF/collagen interaction.

Base Sequence↗
PubMed1990

[Control of the incisor axis in the vestibulo-lingual direction using an original titanium-molybdenum alloy arch].

In order to restore the vestibulo-lingual orientation of the incisor axis while controlling the position of the crowns, the author suggests using a simple original arch, compatible with any of the multi-clip technics used by orthodontists. This arch is made of Titanium-Molybdenum Alloy and presents the distinctive feature of being specially shaped, on the ribbon side, so as to be introduced edgewise. Practical ease, efficiency in obtaining large amplitude, precisely controlled movements, as well as accurate measurement of torque make of this instrument a practical tool for controlling the incisor axis in the vestibulo-lingual orientation, and for improving the I/i angle warranteeing the stability of the results. This arch can be used both in monodisciplinary and multidisciplinary orthodontics, more particularly in surgical-orthodontic management of retrognathia combined with traumatic supraclusion of incisors or with linguoversions of central upper teeth.

Adolescent↗
PubMed1989

Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains.

A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.

Binding Sites↗
PubMed1989

Localization within the 106 N-terminal amino acids of von Willebrand factor (vWF) of the epitope corresponding to a monoclonal antibody which inhibits vWF binding to factor VIII.

We have used an in vitro transcription-translation system to localize the epitope corresponding to a monoclonal antibody (MAb 418) to vWF which specifically inhibits its binding to F. VIII. We have subcloned 1.5 Kb of vWF cDNA encoding for the N-terminal part of the vWF subunit which contains a binding site for F.VIII into the Bluescribe expression vector. After in vitro transcription and translation using the resulting construction (pBS-TG3522), a polypeptide of mol wt 60,000 (AA 1-495) was immunoprecipitated with a polyclonal antibody to vWF and with MAb 418. The MAb 418 epitope was further localized by reducing the size of the cDNA insert. This resulted in the production of two polypeptides of 18,000 (AA 1 to 142) and 13,000 (AA 1 to 106) which both retained reactivity with MAb 418. Thus the epitope corresponding to MAb 418 is localized to the first 106 AA of the mature vWF subunit.

Antibodies, Monoclonal↗
PubMed1989

[Retro-maxillary sequellae of craniofacial stenosis. The value of fronto-facial advancement using the Tessier monobloc technic].

Monobloc fronto-facial advancement (TESSIER's technic) seems to be the best therapeutic to correct retromaxillia, sequellae of cranio-facio-stenosis. From an aesthetical and functional point of view the result is more satisfactory than with a simple LE FORT III osteotomy advancement. Intermaxillary fixation is not necessary. Of course an orthodontic sequence must be associated to correct dental malposition.

Acrocephalosyndactylia↗
PubMed1989

[Constraints of means, technics and results in orthodontics?].

Like all health professionals, Orthodontic Physicians, are constantly seeking or are urged to obtain the best therapeutic results possible by their professional bodies such as Universities, Learned societies, Ordre des Medecins and Professional Associations and by extra-professional bodies such as the Social Security, the Law, the Media and the general Public. The divergence between technical progress and patient care resources is currently creating tension in all western countries; thereby producing difficulties in conditions of practice.

Costs and Cost Analysis↗
PubMed1988

A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII.

We have constructed new B domain deletion derivatives of human factor VIII (FVIII) by manipulating the cDNA using recombinant DNA techniques. One of these new derivatives, FVIII delta II, in which amino acids 771(pro)-1666(asp) have been deleted, no longer contains the protease cleavage site at amino acid position 1648(arg)-1649(glu) known to be involved in the initial step of FVIII processing. We have expressed this molecule in both baby hamster kidney (BHK) 21 cells using the vaccinia virus (VV) expression system and have established Chinese hamster ovary (CHO) derived permanent cell lines expressing either recombinant (r)FVIII or FVIII delta II. The characteristics of FVIII delta II have been compared to those of rFVIII and/or plasma derived (pd) FVIII. FVIII delta II has the following properties: (i) it exhibits FVIII procoagulant activity; (ii) it is expressed at 5-fold higher levels than is the complete molecule in comparable systems; (iii) it migrates for the most part as a single major band on SDS-PAGE, in contrast to the complete molecule; (iv) it is activated to a greater extent by thrombin than is either rFVIII or pdFVIII; and (v) it retains the ability to bind von Willebrand factor (vWf).

Antibodies, Monoclonal↗
PubMed1988

Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe.

To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.

Animals↗